SMYD1 Monoclonal Antibody

What is SMYD1 and why is it a significant research target?

SMYD1 (SET and MYND domain-containing protein 1) is a histone-lysine N-methyltransferase that functions as a transcriptional repressor. It is essential for cardiomyocyte differentiation and cardiac morphogenesis, playing a key role in myogenesis - the process by which muscle cells differentiate and mature. Its critical involvement in muscle formation and maintenance makes it a valuable target for studies on muscular disorders, cardiac diseases, and muscular dystrophies . When designing experiments targeting SMYD1, researchers should consider its dual localization in both cytoplasm and nucleus, as well as its multiple biological functions in chromatin remodeling, transcription regulation, and heart development.

What are the common synonyms and identifiers for SMYD1?

SMYD1 is known by several synonyms in scientific literature and databases:

• BOP (CD8 beta opposite)

• KMT3D

• ZMYND18 (zinc finger, MYND domain containing 18)

• ZMYND22

Important identifier information includes:

• UniProt Code: Q8NB12

• NCBI Gene ID: 150572

• NCBI Accession: Q8NB12.1

• Chromosomal Location: 2p11.2

• Calculated Molecular Weight: 56kDa

• Observed Molecular Weight: 57kDa

Understanding these alternative nomenclatures is essential when conducting literature searches or database queries to ensure comprehensive coverage of SMYD1-related research.

What are the known biological functions of SMYD1?

SMYD1 exhibits multiple biological functions that make it important in developmental and physiological processes:

• Transcriptional repression: Acts as a transcriptional repressor in multiple contexts

• Histone methylation: Functions as a histone-lysine N-methyltransferase (EC 2.1.1.43)

• Cardiac development: Essential for cardiomyocyte differentiation and cardiac morphogenesis

• Muscle development: Critical regulator of myoblast differentiation (positive regulation)

• Inflammatory response: Contributes to LPS-triggered expression and secretion of IL-6 in endothelial cells

• Epigenetic regulation: Affects the H3K4me3 methylation pattern of the IL-6 promoter

When designing experiments to study SMYD1 function, researchers should consider which specific activity they are targeting and select appropriate readouts accordingly.

How does SMYD1 regulate inflammatory responses in endothelial cells?

SMYD1 plays a significant role in inflammatory responses in endothelial cells through several mechanisms:

• LPS stimulation upregulates SMYD1 expression in endothelial cells, as demonstrated in EA.hy926 cells

• SMYD1 contributes to LPS-triggered expression and secretion of IL-6

• SMYD1 induces IL-6 expression through both NF-κB-dependent and NF-κB-independent pathways

• The methyltransferase activity of SMYD1 is directly involved in regulating IL-6 expression

• SMYD1 affects the H3K4me3 (histone H3 lysine 4 trimethylation) pattern at the IL-6 promoter

For researchers investigating this pathway, experimental designs should include methyltransferase activity assays, chromatin immunoprecipitation (ChIP) for H3K4me3 at the IL-6 promoter, and analysis of NF-κB activation status alongside SMYD1 expression manipulation.

What is the relationship between SMYD1's methyltransferase activity and its biological functions?

SMYD1's histone-lysine N-methyltransferase activity is central to many of its biological functions. Research has demonstrated:

• The methyltransferase domain is essential for SMYD1's role in regulating gene expression

• SMYD1 methyltransferase activity directly influences the H3K4me3 methylation pattern at specific gene promoters, including IL-6

• Experiments with methyltransferase-deficient SMYD1 mutants (HMTase mutants) show altered regulatory capacity on target genes

• SMYD1 can affect gene expression through both methyltransferase-dependent and independent mechanisms

When investigating SMYD1 functions, researchers should consider including methyltransferase-dead mutants as controls to distinguish between enzymatic and scaffolding functions of the protein.

What are the subcellular localization patterns of SMYD1 and how do they change upon cellular stimulation?

SMYD1 exhibits dynamic subcellular localization that can be altered by cellular stimulation:

• Under basal conditions, SMYD1 is detected in both cytoplasm and nucleus

• Upon LPS stimulation (1 μg/mL for 3h), SMYD1 shows increased nuclear and cytoplasmic expression in endothelial cells

• Quantitative analysis reveals that LPS stimulation significantly increases SMYD1 immunoreactivity in both nuclear and cytosolic compartments

• This translocation pattern suggests potential distinct functions in different cellular compartments

For proper analysis of SMYD1 localization, researchers should employ both immunocytochemistry and subcellular fractionation followed by immunoblotting to confirm compartment-specific enrichment.

What are the optimal conditions for detecting SMYD1 using immunoblotting techniques?

For optimal detection of SMYD1 using immunoblotting techniques:

• Sample preparation:

  • Total cell lysates should be prepared in a buffer containing protease inhibitors

  • Protein concentration should be determined using Bradford or BCA assay

  • Load 20-40 μg of total protein per lane

• Electrophoresis and transfer:

  • Use 10% SDS-PAGE for optimal separation

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour

• Antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA

  • Use anti-SMYD1 antibody at a dilution of 1:200 to 1:2000

  • Incubate overnight at 4°C for primary antibody

  • Use appropriate HRP-conjugated secondary antibody (e.g., anti-rabbit IgG)

• Detection:

  • SMYD1 is observed at approximately 57 kDa

  • Positive control samples include Raji and MCF7 cell lysates

This protocol can be adjusted based on specific experimental needs and the particular anti-SMYD1 antibody being used.

What are the recommended protocols for immunohistochemistry and immunofluorescence using SMYD1 antibodies?

For immunohistochemistry (IHC) and immunofluorescence (IF) detection of SMYD1:

• Sample preparation:

  • For tissues: Fix in 4% paraformaldehyde, paraffin-embed or freeze, and section (4-6 μm)

  • For cells: Grow on coverslips, fix with 4% paraformaldehyde for 15 minutes at room temperature

• Antigen retrieval (for IHC):

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Microwave for 15-20 minutes or pressure cooker for 3-5 minutes

• Blocking and permeabilization:

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes

  • Block with 5-10% normal serum (from the species of secondary antibody) for 1 hour

• Antibody incubation:

  • For IHC: Anti-SMYD1 antibody at 1:20 to 1:200 dilution

  • For IF: Anti-SMYD1 antibody at 1:20 to 1:100 dilution

  • Incubate overnight at 4°C

• Detection:

  • For IHC: Use appropriate HRP-conjugated secondary antibody and DAB substrate

  • For IF: Use fluorescently-labeled secondary antibodies (e.g., FITC-conjugated anti-rabbit IgG)

  • Counterstain nuclei with DAPI or Draq5

• Recommended positive control tissues:

  • Mouse stomach

  • Rat heart

  • Rat stomach

These protocols should be optimized for specific applications and tissue types.

How can researchers manipulate SMYD1 expression for functional studies?

To effectively manipulate SMYD1 expression for functional studies:

• Overexpression approaches:

  • Transfection with pCMV2-Smyd1-flag vector in cell lines

  • Adenoviral or lentiviral vectors for harder-to-transfect cells

  • Inducible expression systems (e.g., Tet-On) for temporal control

• Knockdown/silencing strategies:

  • Transfection with SMYD1-specific siRNA (validated for efficacy)

  • shRNA delivered via lentiviral vectors for stable knockdown

  • CRISPR-Cas9 system for gene knockout studies

• Verification of manipulation:

  • RT-qPCR to quantify SMYD1 mRNA levels

  • Immunoblotting to confirm protein level changes

  • Functional assays to validate consequences of expression changes

• Experimental controls:

  • Empty vector controls for overexpression studies

  • Non-targeting siRNA/shRNA for knockdown studies

  • Wild-type cells alongside CRISPR-modified cells

These approaches have been validated in endothelial cell models and can be adapted for other cell types depending on transfection efficiency.

How should researchers address inconsistent SMYD1 antibody staining patterns?

When encountering inconsistent SMYD1 antibody staining patterns:

• Antibody validation:

  • Confirm antibody specificity using positive and negative control samples

  • Validate with knockdown/knockout samples to ensure specificity

  • Consider using multiple antibodies targeting different epitopes

• Protocol optimization:

  • Test multiple fixation methods (paraformaldehyde, methanol, acetone)

  • Optimize antigen retrieval conditions (pH, duration, temperature)

  • Titrate antibody concentration to determine optimal dilution

  • Extend primary antibody incubation time (overnight at 4°C)

• Signal enhancement:

  • Use signal amplification systems (e.g., tyramide signal amplification)

  • Optimize exposure settings for imaging

  • Consider using alternative detection systems

• Background reduction:

  • Increase blocking time and concentration

  • Add 0.1-0.3% Triton X-100 to antibody diluent

  • Include additional washing steps with higher salt concentration

• Subcellular localization considerations:

  • SMYD1 is present in both nuclear and cytoplasmic compartments

  • Different fixation methods may preferentially preserve one compartment

  • Consider subcellular fractionation for Western blot analysis

Documenting all optimization steps will help establish reproducible protocols for future experiments.

How can researchers distinguish between SMYD1 isoforms and related SMYD family proteins?

To accurately distinguish between SMYD1 isoforms and related SMYD family proteins:

• Isoform identification:

  • Use isoform-specific primers for RT-PCR

  • Employ antibodies targeting isoform-specific regions

  • Analyze molecular weight differences on Western blots (56-57 kDa for SMYD1)

• Family member discrimination:

  • Select antibodies validated for lack of cross-reactivity with other SMYD family members

  • Include positive controls for each SMYD family member

  • Use siRNA knockdown of specific SMYD proteins as negative controls

• Sequence analysis considerations:

  • SMYD1 shares sequence similarity with other SMYD family members

  • The SET domain is highly conserved across the family

  • The MYND domain provides some distinction between family members

• Expression pattern analysis:

  • SMYD1 has distinctive tissue expression patterns (heart, skeletal muscle)

  • Compare expression patterns with known profiles of other SMYD proteins

  • Use tissue-specific controls when available

Combining multiple approaches provides the most reliable discrimination between closely related proteins.

What are the key considerations when analyzing SMYD1's role in inflammatory responses?

When analyzing SMYD1's role in inflammatory responses, researchers should consider:

• Stimulus-specific effects:

  • LPS concentration-dependent effects (1 ng/mL to 10 μg/mL)

  • Time-dependent changes (short-term vs. long-term exposure)

  • Different inflammatory stimuli may yield varying responses

• Cell type considerations:

  • Endothelial cells (such as EA.hy926) show specific responses

  • Primary vs. immortalized cell differences

  • Tissue-specific endothelial cells may respond differently

• Pathway analysis:

  • NF-κB-dependent mechanisms

  • NF-κB-independent mechanisms

  • Interaction with other inflammatory signaling pathways

• Epigenetic analysis:

  • H3K4me3 modifications at specific promoters

  • Changes in DNA accessibility

  • Integration with transcription factor binding

• Downstream effects measurement:

  • IL-6 expression and secretion

  • Other inflammatory cytokines

  • Functional consequences on cell behavior

A comprehensive analysis should include both molecular mechanisms and functional outcomes to fully characterize SMYD1's role in inflammation.

What are the emerging applications of SMYD1 research in cardiovascular disease models?

SMYD1's essential role in cardiac development suggests several promising research directions:

• Cardiac regeneration:

  • SMYD1's potential role in cardiac progenitor cell differentiation

  • Application in directing stem cell fate toward cardiomyocyte lineage

  • Development of SMYD1-targeting strategies to enhance cardiac repair

• Heart failure models:

  • Changes in SMYD1 expression and activity in different heart failure models

  • Correlation with cardiac remodeling and fibrosis

  • Potential therapeutic targeting to mitigate adverse remodeling

• Inflammatory cardiac conditions:

  • SMYD1's contribution to inflammatory signaling in cardiac tissue

  • Role in myocarditis and other inflammatory heart diseases

  • Interaction with cardiac immune cell populations

• Developmental cardiac defects:

  • SMYD1 mutations in congenital heart disease

  • Epigenetic profiling of SMYD1-regulated genes during cardiogenesis

  • Model organism studies of SMYD1 in heart development

These research areas may yield new insights into cardiac pathophysiology and identify novel therapeutic strategies.

How can multi-omics approaches enhance our understanding of SMYD1 function?

Integrating multi-omics approaches can provide comprehensive insights into SMYD1 function:

• Epigenomic approaches:

  • ChIP-seq for genome-wide mapping of SMYD1 binding sites

  • CUT&RUN for higher resolution protein-DNA interaction mapping

  • ATAC-seq to correlate SMYD1 activity with chromatin accessibility changes

• Transcriptomic analysis:

  • RNA-seq following SMYD1 manipulation to identify regulated gene networks

  • Single-cell RNA-seq to capture cell-specific responses

  • Nascent RNA analysis to distinguish direct from indirect effects

• Proteomic strategies:

  • IP-MS to identify SMYD1 protein interaction partners

  • Phosphoproteomics to map signaling cascades affected by SMYD1

  • Proteome-wide analysis of histone modifications

• Metabolomic considerations:

  • Analysis of metabolic changes in SMYD1-manipulated systems

  • Connection between cellular metabolism and SMYD1 activity

  • SAM (S-adenosyl methionine) availability effects on SMYD1 function

• Integrated data analysis:

  • Network analysis to identify SMYD1-regulated hubs

  • Machine learning approaches to predict SMYD1 targets

  • Temporal multi-omics to track dynamic responses

These approaches can reveal previously unrecognized functions and regulatory mechanisms of SMYD1.

What are the key specifications for selecting appropriate SMYD1 antibodies?

When selecting SMYD1 antibodies for research applications, consider these technical specifications:

Always validate antibodies in your specific experimental system before conducting critical experiments.

What are the recommended experimental conditions for studying SMYD1's methyltransferase activity?

For studying SMYD1's methyltransferase activity, the following experimental conditions are recommended:

• Enzyme preparation:

  • Recombinant SMYD1 expression in E. coli or baculovirus systems

  • FLAG-tagged or His-tagged protein for purification

  • Active enzyme requires proper folding and possible co-factors

• Assay conditions:

  • Buffer: 50 mM Tris-HCl (pH 8.0), 10% glycerol, 20 mM KCl, 5 mM MgCl₂

  • Temperature: 30°C is optimal for enzymatic activity

  • Incubation time: 30-60 minutes for standard assays

• Substrates:

  • S-adenosyl methionine (SAM) as methyl donor

  • Recombinant histones (especially H3)

  • Synthetic histone peptides containing target lysine residues

• Activity detection methods:

  • Radioactive assay using ³H-SAM

  • Antibody-based detection of methylated histones

  • Mass spectrometry to identify methylation sites

  • Fluorescence-based assays for high-throughput screening

• Controls:

  • Methyltransferase-dead mutant (HMTase mutant)

  • S-adenosyl homocysteine (SAH) as competitive inhibitor

  • Known methyltransferase inhibitors as reference

These conditions should be optimized for specific experimental objectives and available resources.

How does SMYD1 function compare to other SMYD family proteins?

The SMYD protein family shares structural similarities but exhibits distinct functional properties:

This comparative analysis highlights the unique role of SMYD1 in muscle development and function, distinguishing it from other family members that have broader expression patterns and functions.

How should researchers interpret contradictory results when studying SMYD1 in different experimental systems?

When faced with contradictory results in SMYD1 research across different experimental systems, consider these interpretative frameworks:

• Cell type-specific effects:

  • SMYD1 may have different binding partners in different cell types

  • Chromatin landscape varies between cell types, affecting SMYD1 target accessibility

  • Signal transduction pathways may differ, altering SMYD1 regulation

• Experimental condition variables:

  • Culture conditions (serum levels, oxygen tension, confluency)

  • Stimulus concentrations and duration (e.g., LPS dosage)

  • Acute vs. chronic manipulation of SMYD1 expression

• Methodological considerations:

  • Antibody specificity issues

  • Detection method sensitivity

  • Overexpression artifacts vs. physiological levels

• Isoform-specific effects:

  • Different SMYD1 isoforms may have distinct functions

  • Expression ratios of isoforms may vary between systems

  • Post-translational modifications may differ

• Integration approach:

  • Map contradictions to specific variables

  • Design experiments to directly test conflicting results

  • Consider using multiple complementary techniques

  • Obtain biological replicates across different conditions